Since the discovery of human T-lymphotropic virus (HTLV)-I and its simian counterpart, simian T-lymphotropic virus (STLV)-I, the possible existence of other related simian T-lymphotropic retroviruses has remained active. In our previous report, we described the use of a previously developed HTLV-I recombinant env protein, rpBI, and HTLV-II recombinant env protein, rpBII, in differential immunoblot analyses, in conjunction with the polymerase chain reaction (PCR) technique, to isolate HTLV type II-related viruses from New World monkeys, namely, the spider monkey. A virus was isolated that showed both immunological cross-reactivity and PCR amplification patterns suggestive of a STLV type II. We now show that, by limited nucleotide sequence analysis, the X region of the New World monkey isolate (STLV-II-SM3) reveals a 96.5%, 81.6%, and 77.2% homology with the corresponding X region of prototypic HTLV-II, HTLV-I, and STLV-I, respectively. Ten env gene sequences from human immunodeficiency virus (HIV)-1 isolates obtained from a laboratory worker accidentally infected with the virus were compared to 16 other HIV-1 sequences, as well as the sequence of an HIV-1-like virus isolated from a chimpanzee. The isolates analyzed were derived from blood drawn in the years 1985, 1987, 1988, and 1990. A matrix representing the Jukes-Cantor distances was used to generate a phylogenetic tree by the neighbor-joining method. The robustness of the tree was analyzed by 100 bootstrap resamplings of the original data. A close agreement was found between the topology of the neighbor-joining tree, derived from the original data set, and the consensus tree, derived from the bootstrap replicas. This analysis confirms that the lab worker was infected by a virus from the HTLV-III/LAV group, strongly supporting the hypothesis that the lab worker was infected with the cultured virus that he/she was working with.